Peroxynitrite has potent pulmonary vasodilator activity in the rat

Peroxynitrite (PN) worsens pathological conditions associated with oxidative stress. However, beneficial effects have also been reported. PN has been shown to demonstrate vasodilator as well as vasoconstrictor properties that are dependent upon the experimental conditions and the vascular bed studied. PN-induced vascular smooth muscle relaxation may involve the formation of nitric oxide (NO) donors. The present results show that PN has significant vasodilator activity in the pulmonary and systemic vascular beds, and that responses to PN were not attenuated by L-penicillamine (L-PEN), a PN scavenger, whereas responses to sodium nitroprusside (SNP) were decreased. PN had a small inhibitory effect on decreases in arterial pressure in response to the NO donors diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) and S-nitrosoglutathione (GSNO). PN partially reversed hypoxic pulmonary vasoconstriction. PN responses were attenuated by the soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and responses to PN and the PN precursor, 3-morpholinosydnonimine (SIN-1), were different. These data show that PN has potent pulmonary vasodilator activity in the rat, and provide evidence that a PN interaction with S-nitrosothiols is not the major mechanism mediating the response. These data suggest that responses to PN are mediated by the activation of sGC, and that PN has a small inhibitory effect on NO responses.     

Mechanisms underlying the relaxation induced by isokaempferide from Amburana cearensis in the guinea-pig isolated trachea

The present study examines possible mechanisms by which the flavonoid isokaempferide (IKPF; 5,7,4'-trihydroxy-3-methoxyflavone) from Amburana cearensis, a Brazilian medicinal plant popularly used as bronchodilator, induces relaxation of guinea-pig isolated trachea. In the trachea (with intact epithelium) contracted by carbachol, IKPF (1-1000 microM) caused a graded relaxation, and the epithelium removal increased the sensitivity of the airway smooth muscle to IKPF (EC50, in intact tissue: 77.4 [54.8-109.2] microM; in denuded epithelium: 15.0 [11.3-20.1] microM). The IKPF-induced relaxation was inhibited in 41% by the nitric oxide (NO) synthase inhibitor L-NAME (100 microM); in 31% and 50% by the soluble guanylate cyclase (sGC) inhibitor ODQ (3 and 33 microM); by propranolol (31%) and also by capsaicin (37%). In the trachea pre-contracted by 40 mM KCl the pre-incubation with glibenclamide (33 microM) or iberiotoxin (IbTX, 0.1 microM), selective K(+) channel inhibitors, inhibited the IKPF-induced relaxation by 39% and 38%, respectively. On the other hand, 4-aminopyridine (100 microM), a nonselective K(+) channel antagonist, did not significantly influence the effect of IKPF, while IbTX induced a rightward displacement of the IKPF concentration-response curve. However, in muscle pre-contracted with 120 mM KCl the relaxant effect of IKPF was significantly reduced and not affected by glibenclamide. In conclusion, these results indicate a direct and epithelium-independent relaxant effect of IKPF on smooth muscle fibers. Although this IKPF relaxant action seems to be multi-mediated, it occurs via both Ca(2+) and ATP-sensitive K(+) channels, but some other possible mechanisms unrelated to K(+) channels cannot be excluded.     

Actions of NO donors and endogenous nitrergic transmitter on the longitudinal muscle of rat ileum in vitro: mechanisms involved

The aim of this work has been to characterize and to compare the responses of the rat ileal longitudinal muscle to the nitric oxide (NO) donors, sodium nitroprusside (SNP) and morpholinosydnonimine hydrochloride (SIN-1). SNP (10(-5)-10(-3) M) caused a contraction followed by a relaxation, both components being concentration-dependent. In contrast, SIN-1 (10(-5)-10(-4) M) caused a relaxation followed by a contraction. Neither the neural blocker tetrodotoxin (TTX) nor atropine were able to change the response to SNP, whereas nifedipine abolished its contractile component. In contrast, TTX and nifedipine diminished both the relaxation and the contraction in response to SIN-1, whereas atropine decreased only the contractile component. The specific guanylate cyclase inhibitor oxadiazolo-quinoxalin-1-one (ODQ) decreased the relaxation induced by SNP but did not modify that caused by SIN-1. The K+ channel blockers charybdotoxin, apamin and tetraethylamonium were unable to modify the response to SNP. In contrast, both TEA and apamin significantly decreased the relaxation induced by SIN- 1. The relaxation resulting from electrical field stimulation (EFS) of enteric nerves in non-adrenergic non-cholinergic conditions is mainly but not exclusively nitrergic, as incubation with the NO synthase inhibitor L-NNA markedly decreases such relaxation. EFS-induced relaxation is also sensitive to ODQ. We conclude that SNP acts mainly on smooth muscle cells activating L-type Ca2+ channels, which result in contraction, and activates the soluble guanylate cyclase, which results in relaxation. In contrast SIN-1 has mixed--neuronal and muscular--effects, the contraction being caused both by acetylcholine release from neurons and by direct activation of L-type Ca2+ channels on smooth muscle cells. SIN-1-induced relaxation is cGMP-independent and it is likely to occur as a consequence of both, neuronal release of inhibitory transmitter(s) and by activation of apamin sensitive K+ channels. The effect of the nitrergic transmitter released from enteric nerves is different from those caused by SIN-1 but shows similarities with those caused by SNP.     

Role of conformational changes in the heme-dependent regulation of human soluble guanylate cyclase.

Soluble guanylate cyclase (sGC) is a receptor for endogenous and exogenous nitric oxide (NO) and is activated many fold upon its binding, making it a core enzyme in the nitric oxide signal transduction pathway. Much effort has been made to understand the link between binding of NO at the sGC heme and activation of the cyclase activity. We report here the first direct evidence for the role of conformational changes in transmitting the signal between the heme and cyclase domains. Using both circular dichroism (CD) and fluorescence spectroscopies, we have probed the effect that the sGC activators NO and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazole (YC-1) and the inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ) have on the structure of the protein. Surprisingly, binding of either ODQ or YC-1 to NO-bound sGC cause virtually identical changes in the far-UV CD spectra of sGC, reflecting a perturbation in the secondary structure of the enzyme. This change is absent upon binding of NO, YC-1 or ODQ alone. Using this and previous data, we propose a working model for the mechanism of activation of sGC by NO and YC-1 and inhibition by ODQ.     

Ursolic acid mediates the vasorelaxant activity of Lepechinia caulescens via NO release in isolated rat thoracic aorta

We have determined that the methanolic extract of L. caulescens (MELc) produced a significant vasodilator effect in a concentration-dependent and endothelium-dependent manner. This relaxation was blocked by N(omega)-nitro-L-arginine methylester (L-NAME), indicating that MELc vasodilator properties are endothelium mediated due to liberation of nitric oxide (NO). In this paper we aimed to corroborate its mode of action. MELc effects on noradrenaline (NA)-induced contraction in isolated rat aortic thoracic rings with endothelium (+E), in the presence of atropine (0.1 microM) and 1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ, 1 microM) were conducted. MELc relaxation curve was significantly shifted to the right in the presence of ODQ and atropine, thus confirming that its mode of action is related with activation of nitric oxide synthase (NOS) and the consequent increment in NO formation. Bio-guided study of MELc allowed the isolation of ursolic acid (UA, 50 mg) and ursolic-oleanolic acids mixture [UA/OA (7:3), 450 mg]. The relaxant effect of UA (0.038-110 microM) was evaluated in functional experiments. UA induced a significant relaxation in a concentration- and endothelium-dependent manner (IC(50)=44.15 microM) and did not produce a vasorelaxant effect on contraction evoked by KCl (80 mM). In addition, NA-induced contraction was significantly displaced to the right by UA (30 microM). In order to determine its mode of action, UA-induced relaxant effect was evaluated in the presence of atropine (0.1 microM), indomethacin (10 microM), L-NAME (100 microM) and ODQ (1 microM). Relaxation was blocked by L-NAME and ODQ. On the other hand, UA (3 microM) provoked a significant displacement to the left in the relaxation curve induced by sodium nitroprusside (SNP, 0.32 nM to 0.1 microM), but it was not significant in the presence of Carbamoyl choline (carbachol, 1 nM to 10 microM). These results indicate that UA-mediated relaxation is endothelium dependent, probably due to NO release, and the consequent activation of vascular smooth muscle soluble guanylate cyclase (sGC), a signal transduction enzyme that forms the second messenger cGMP.     

Role of cGMP as a mediator of nerve-induced motor functions of the opossum esophagus

Stimulation of esophageal nerves produces biphasic relaxation of the lower esophageal sphincter (LES) and an off response of circular esophageal muscle. Previously, we proposed that cGMP mediates nerve-induced hyperpolarization of circular LES muscle but not LES relaxation. These experiments explore whether cGMP mediates LES relaxation or the off response. Strips of muscle from the opossum esophagus and LES were connected to force-displacement transducers, placed in tissue baths containing oxygenated Krebs solution at 37 degrees C, and stimulated by an electrical field. 1H-[1,2, 4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of guanylyl cyclase, antagonized the off response, shortened its latency, and blocked the first phase of LES relaxation. ODQ also antagonized LES relaxation by exogenous nitric oxide (NO) but not relaxations by vasoactive intestinal polypeptide (VIP). Part of the nerve-induced LES relaxation and the off response appear to be mediated by the second messenger cGMP. These studies indicate that VIP-induced LES relaxation is not mediated by cGMP and therefore do not support the hypothesis that VIP produces LES relaxation by causing the generation of NO.     

Essential role of nitric oxide in acute ischemic preconditioning: S-Nitros(yl)ation versus sGC/cGMP/PKG signaling?

Nitric oxide (NO) plays an important role in acute ischemic preconditioning (IPC). In addition to activating soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signaling pathways, NO-mediated protein S-nitros(yl)ation (SNO) has been recently shown to play an essential role in cardioprotection against ischemia–reperfusion (I/R) injury. In our previous studies, we have shown that IPC-induced cardioprotection could be blocked by treatment with either N-nitro-L-arginine methyl ester (L-NAME, a constitutive NO synthase inhibitor) or ascorbate (a reducing agent to decompose SNO). To clarify NO-mediated sGC/cGMP/PKG-dependent or -independent (i.e., SNO) signaling involved in IPC-induced cardioprotection, mouse hearts were Langendorff-perfused in the dark to prevent SNO decomposition by light exposure. Treatment with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, a highly selective inhibitor of sGC) or KT5823 (a potent and selective inhibitor of PKG) did not abolish IPC-induced acute protection, suggesting that the sGC/cGMP/PKG signaling pathway does not play an important role in NO-mediated cardioprotective signaling during acute IPC. In addition, treatment with ODQ in IPC hearts provided an additional protective effect on functional recovery, in parallel with a higher SNO level in these ODQ+IPC hearts. In conclusion, these results suggest that the protective effect of NO is not related primarily to activation of the sGC/cGMP/PKG signaling pathway, but rather through SNO signaling in IPC-induced acute cardioprotection.     

Endothelium-dependent and -independent vasorelaxation by a theophylline derivative MCPT: roles of cyclic nucleotides, potassium channel opening and phosphodiesterase inhibition

The vasorelaxation activities of MCPT, a newly synthesized xanthine derivative, were investigated in this study. In phenylephrine (PE)-precontracted rat aortic rings with intact endothelium, MCPT caused a concentration-dependent relaxation, which was inhibited by endothelium removed. This relaxation was also reduced by the presence of nitric oxide synthase inhibitor Nomega-nitro-L-arginine methylester (L-NAME, 100 microM), soluble guanylyl cyclase (sGC) inhibitors methylene blue (10 microM), 1 H-[1,2,4] oxidazolol [4,3-a] quinoxalin-1-one (ODQ, 1 microM), adenylyl cyclase (AC) blocker SQ 22536 (100 microM), ATP-sensitive K+ channel blocker (KATP) glibenclamide (1 microM), a Ca2+ activated K+ channels blocker tetraethylammonium (TEA, 10 mM) and a voltage-dependent potassium channels blocker 4-aminopyridine (4-AP, 100 microM). The vasorelaxant effects of MCPT together with IBMX (0.5 microM) had an additive action. In PE-preconstricted endothelium-denuded aortic rings, the vasorelaxant effects of MCPT were attenuated by pretreatments with glibenclamide (1 microM), SQ 22536 (100 microM) or ODQ (1 microM), respectively. MCPT enhanced cAMP-dependent vasodilator isoprenaline- and NO donor/cGMP-dependent vasodilator sodium nitroprusside-induced relaxation activities in endothelium-denuded aortic rings. In A-10 cell and washed human platelets, MCPT induced a concentration-dependent increase in intracellular cyclic GMP and cyclic AMP levels. In phosphodiesterase assay, MCPT displayed inhibition effects on PDE 3, PDE 4 and PDE 5. The inhibition % were 52 +/- 3.9, 32 +/- 2.6 and 8 +/- 1.1 respectively. The Western blot analysis on HUVEC indicated that MCPT increased the expression of eNOS. It is concluded that the vasorelaxation by MCPT may be mediated by the inhibition of phosphodiesterase, stimulation of NO/sGC/ cGMP and AC/cAMP pathways, and the opening of K+ channels.     

Modulation of nitrergic relaxant responses by peptides in the mouse gastric fundus

The effects of pituitary adenylate cyclase-activating peptide (PACAP-38) and vasoactive intestinal polypeptide (VIP) were investigated in the gastric fundus strips of the mouse. In carbachol (CCh) precontracted strips, in the presence of guanethidine, electrical field stimulation (EFS) elicited a fast inhibitory response that may be followed, at the highest stimulation frequencies employed, by a sustained relaxation. The fast response was abolished by the nitric oxide (NO) synthesis inhibitor L-N(G)-nitro arginine (L-NNA) or by the guanylate cyclase inhibitor (ODQ), the sustained one by alpha-chymotrypsin. alpha-Chymotrypsin also increased the amplitude of the EFS-induced fast relaxation. PACAP-38 and VIP caused tetrodotoxin-insensitive sustained relaxant responses that were both abolished by alpha-chymotrypsin. Apamin did not influence relaxant responses to EFS nor relaxation to both peptides. PACAP 6-38 abolished EFS-induced sustained relaxations, increased the amplitude of the fast ones and antagonized the smooth muscle relaxation to both PACAP-38 and VIP. VIP 10-28 and [D-p-Cl-Phe6,Leu17]-VIP did not influence the amplitude of both the fast or the sustained response to EFS nor influenced the relaxation to VIP and PACAP-38. The results indicate that in strips from mouse gastric fundus peptides, other than being responsible for EFS-induced sustained relaxation, also exerts a modulatory action on the release of the neurotransmitter responsible for the fast relaxant response, that appears to be NO.     

Protoporphyrin IX generation from delta-aminolevulinic acid elicits pulmonary artery relaxation and soluble guanylate cyclase activation

Protoporphyrin IX is an activator of soluble guanylate cyclase (sGC), but its role as an endogenous regulator of vascular function through cGMP has not been previously reported. In this study we examined whether the heme precursor delta-aminolevulinic acid (ALA) could regulate vascular force through promoting protoporphyrin IX-elicited activation of sGC. Exposure of endothelium-denuded bovine pulmonary arteries (BPA) in organoid culture to increasing concentrations of the heme precursor ALA caused a concentration-dependent increase in BPA epifluorescence, consistent with increased tissue protoporphyrin IX levels, associated with decreased force generation to increasing concentrations of serotonin. The force-depressing actions of 0.1 mM ALA were associated with increased cGMP-associated vasodilator-stimulated phosphoprotein (VASP) phosphorylation and increased sGC activity in homogenates of BPA cultured with ALA. Increasing iron availability with 0.1 mM FeSO(4) inhibited the decrease in contraction to serotonin and increase in sGC activity caused by ALA, associated with decreased protoporphyrin IX and increased heme. Chelating endogenous iron with 0.1 mM deferoxamine increased the detection of protoporphyrin IX and force depressing activity of 10 microM ALA. The inhibition of sGC activation with the heme oxidant 10 muM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) attenuated the force depressing actions of an NO donor without altering the actions of ALA. Thus control of endogenous formation of protoporphyrin IX from ALA by the availability of iron is potentially a novel physiological mechanism of controlling vascular function through regulating the activity of sGC.     

Potentiation of carbon monoxide-induced relaxation of rat aorta by YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole

The hypothesis that endogenous carbon monoxide (CO), produced during the oxidation of heme catalyzed by heme oxygenase (HO), plays a role similar to that of nitric oxide (NO) in the regulation of cardiovascular tone has been criticized because of the low potency of CO compared with NO in relaxing blood vessels and stimulating soluble guanylyl cyclase (sGC). This criticism has been muted by the demonstration that, in the presence of YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole], CO has similar potency to NO in stimulating sGC activity. In this study, we determined that YC-1 potentiated CO-induced relaxation of rat aortic strips (RtAS) by approximately ten-fold. Furthermore, CO-induced relaxation of RtAS was shown to be mediated through stimulation of sGC because vasorelaxation was inhibited by ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one), a selective sGC inhibitor, in the absence and presence of YC-1. A gas chromatographic-headspace method was used to measure CO concentration in Krebs' solution following the addition of CO-saturated saline solution to the tissue bath, in order to provide an accurate determination of RtAS exposure to CO. The tissue bath concentration of CO was shown to be approximately one-half of that calculated to be present. We conclude that should an endogenous compound exist with properties similar to that of YC-1, then the potency of CO as a vasorelaxant in the presence of this factor would be increased. As a consequence, CO could play a role in the regulation of cardiovascular tone, comparable to that of NO.     

Adenosine/nitric oxide crosstalk in the branchial circulation of Squalus acanthias and Anguilla anguilla

The potent vasomodulator adenosine (AD), thanks to the interaction with by A(1) and A(2) receptors, dilates systemic, coronary and cerebral vasculatures but exert a constrictor action in several vessels of respiratory organs. Recent investigations suggest that nitric oxide (NO) contributes to AD effects. In fish, both NO and AD induce atypical effects compared to mammals. Since there is very little information on the role of NO and its involvement in mediating the actions of AD in fish, we have analysed this question in the branchial vasculature of the elasmobranch Squalus acanthias and the teleost Anguilla anguilla using an isolated perfused head and a branchial basket preparation, respectively. In both dogfish and eel, AD dose-response curves showed a biphasic effect: vasoconstriction (pico to nanomolar range) and vasodilation (micromolar range). Both effects were abolished by the classic xanthine inhibitor theophylline (Theo) and also by specific antagonists of A(1) and A(2) receptor subtypes. To analyse the involvement of the NO/cGMP system in the AD responses, we tested a NOS inhibitor, l-NIO, and a specific soluble guanylate cyclase (sGC) blocker, ODQ. In both dogfish and eel preparations l-NIO abrogated all vasomotor effects of AD, whereas ODQ blocked the AD-mediated vasoconstriction without affecting the vasorelaxant response. This indicates that only AD-induced vasoconstriction is mediated by a NO-cGMP-dependent mechanism. By using the NO donor SIN-1, we showed a dose-dependent vasoconstrictory effect which was completely blocked by ODQ. These results provide compelling evidence that the vasoactive role of AD in the branchial circulation of S. acanthias and A. anguilla involves a NO signalling.     

Peroxynitrite enhances astrocytic volume-sensitive excitatory amino acid release via a src tyrosine kinase-dependent mechanism

Volume-regulated anion channels (VRACs) are critically important for cell volume homeostasis, and under pathological conditions contribute to neuronal damage via excitatory amino (EAA) release. The precise mechanisms by which brain VRACs are activated and/or modulated remain elusive. In the present work we explored the possible involvement of nitric oxide (NO) and NO-related reactive species in the regulation of VRAC activity and EAA release, using primary astrocyte cultures. The NO donors sodium nitroprusside and spermine NONOate did not affect volume-activated d-[3H]aspartate release. In contrast, the peroxynitrite (ONOO-) donor 3-morpholinosydnomine hydrochloride (SIN-1) increased volume-dependent EAA release by approx. 80-110% under identical conditions. Inhibition of ONOO- formation with superoxide dismutase completely abolished the effects of SIN-1. Both the volume- and SIN-1-induced EAA release were sensitive to the VRAC blockers NPPB and ATP. Further pharmacological analysis ruled out the involvement of cGMP-dependent reactions and modification of sulfhydryl groups in the SIN-1-inducedmodulation of EAA release. The src family tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine (PP2), but not its inactive analog PP3, abolished the effects of SIN-1. A broader spectrum tyrosine kinase inhibitor tyrphostin A51, also completely eliminated the SIN-1-induced EAA release. Our data suggest that ONOO- up-regulates VRAC activity via a src tyrosine kinase-dependent mechanism. This modulation may contribute to EAA-mediated neuronal damage in ischemia and other pathological conditions favoring cell swelling and ONOO- production.     

Investigation of the vasorelaxant effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) and diethylamine/nitric oxide (DEA/NO) on the human radial artery used as coronary bypass graft

The radial artery (RA) is used as a spastic coronary bypass graft. This study was designed to investigate the mechanism of vasorelaxant effects of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole), a nitric oxide (NO)-independent soluble guanylate cyclase (sGC) activator, and DEA/NO (diethylamine/nitric oxide), a NO-nucleophile adduct, on the human RA. RA segments (n = 25) were obtained from coronary artery bypass grafting patients and were divided into 3-4 mm vascular rings.Using the isolated tissue bath technique, the endothelium-independent vasodilatation function was tested in vitro by the addition of cumulative concentrations of YC-1 (10-10 to 3 x 10-7 mol/L) and DEA/NO (10-8 to 3 x 10-5 mol/L) following vasocontraction by phenylephrine in the presence or absence of 10-5 mol/L ODQ (1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one), the selective sGC inhibitor, 10-7 mol/L iberiotoxin, a blocker of Ca2+-activated K+ channels, or 10-5 mol/L ODQ plus 10-7 mol/L iberiotoxin. We also evaluated the effect of YC-1 and DEA/NO on the cGMP levels in vascular rings obtained from human radial artery (n = 6 for each drug). YC-1 (10-10 to 3 x 10-7 mol/L) and DEA/NO (10-8 to 3 x 10-5 mol/L) caused the concentration-dependent vasorelaxation in RA rings precontracted with phenylephrine (10-5 mol/L) (n = 20 for each drug). Pre-incubation of RA rings with ODQ, iberiotoxin, or ODQ plus iberiotoxin significantly inhibited the vasorelaxant effect of YC-1, but the inhibitor effect of ODQ plus iberiotoxin was significantly more than that of ODQ and iberiotoxin alone (p < 0.05). The vasorelaxant effect of DEA/NO almost completely abolished in the presence of ODQ and iberiotoxin plus ODQ, but did not significantly change in the presence of iberiotoxin alone (p > 0.05). The pEC50 value of DEA/NO was significantly lower than those for YC-1 (p < 0.01), with no change Emax values in RA rings. In addition, YC-1-stimulated RA rings showed more elevation in cGMP than that of DEA/NO (p < 0.05). These findings indicate that YC-1 is a more potent relaxant than DEA/NO in the human RA. The relaxant effects of YC-1 could be due to the stimulation of the sGC and Ca2+-sensitive K+channels, whereas the relaxant effects of DEA/NO could be completely due to the stimulation of the sGC. YC-1 and DEA/NO may be effective as vasodilator for the short-term treatment of perioperative spasm of coronary bypass grafts.     

Inhibition of soluble guanylate cyclase by ODQ

The heme in soluble guanylate cyclases (sGC) as isolated is ferrous, high-spin, and 5-coordinate. [1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one] (ODQ) has been used extensively as a specific inhibitor for sGC and as a diagnostic tool for identifying a role for sGC in signal transduction events. Addition of ODQ to ferrous sGC leads to a Soret shift from 431 to 392 nm and a decrease in nitric oxide (NO)-stimulated sGC activity. This Soret shift is consistent with oxidation of the ferrous heme to ferric heme. The results reported here further define the molecular mechanism of inhibition of sGC by ODQ. Addition of ODQ to the isolated sGC heme domain [beta1(1-385)] gave the same spectral changes as when sGC was treated with ODQ. EPR and resonance Raman spectroscopy was used to show that the heme in ODQ-treated beta1(1-385) is indeed ferric. Inhibition of the NO-stimulated sGC activity by ODQ is due to oxidation of the sGC heme and not to perturbation of the catalytic site, since the ODQ-treated sGC has the same basal activity as untreated sGC (68 +/- 12 nmol min(-)(1) mg(-)(1)). In addition, ODQ-oxidized sGC can be re-reduced by dithionite, and this re-reduced sGC has identical NO-stimulated activity as the original ferrous sGC. Oxidation of the sGC heme by ODQ is fast with a second-order rate constant of 8.5 x 10(3) M(-)(1) s(-)(1). ODQ can also oxidize hemoglobin, indicating that the reaction is not specific for the heme in sGC versus that in other hemoproteins.     

Antagonism of soluble guanylyl cyclase attenuates cutaneous vasodilation during whole body heat stress and local warming in humans

We hypothesized that nitric oxide activation of soluble guanylyl cyclase (sGC) participates in cutaneous vasodilation during whole body heat stress and local skin warming. We examined the effects of the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), on reflex skin blood flow responses to whole body heat stress and on nonreflex responses to increased local skin temperature. Blood flow was monitored by laser-Doppler flowmetry, and blood pressure by Finapres to calculate cutaneous vascular conductance (CVC). Intradermal microdialysis was used to treat one site with 1 mM ODQ in 2% DMSO and Ringer, a second site with 2% DMSO in Ringer, and a third site received Ringer. In protocol 1, after a period of normothermia, whole body heat stress was induced. In protocol 2, local heating units warmed local skin temperature from 34 to 41°C to cause local vasodilation. In protocol 1, in normothermia, CVC did not differ among sites [ODQ, 15 ± 3% maximum CVC (CVC(max)); DMSO, 14 ± 3% CVC(max); Ringer, 17 ± 6% CVC(max); P > 0.05]. During heat stress, ODQ attenuated CVC increases (ODQ, 54 ± 4% CVC(max); DMSO, 64 ± 4% CVC(max); Ringer, 63 ± 4% CVC(max); P < 0.05, ODQ vs. DMSO or Ringer). In protocol 2, at 34°C local temperature, CVC did not differ among sites (ODQ, 17 ± 2% CVC(max); DMSO, 18 ± 4% CVC(max); Ringer, 18 ± 3% CVC(max); P > 0.05). ODQ attenuated CVC increases at 41°C local temperature (ODQ, 54 ± 5% CVC(max); DMSO, 86 ± 4% CVC(max); Ringer, 90 ± 2% CVC(max); P < 0.05 ODQ vs. DMSO or Ringer). sGC participates in neurogenic active vasodilation during heat stress and in the local response to direct skin warming.     

Calcium/calmodulin-dependent kinase II mediates NO-elicited PKG activation to participate in spinal reflex potentiation in anesthetized rats

Calcium/calmodulin protein kinase (CaMK)-dependent nitric oxide (NO) and the downstream intracellular messenger cGMP, which is activated by soluble guanylate cyclase (sGC), are believed to induce long-term changes in efficacy of synapses through the activation of protein kinase G (PKG). The aim of this study was to examine the involvement of the CaMKII-dependent NO/sGC/PKG pathway in a novel form of repetitive stimulation-induced spinal reflex potentiation (SRP). A single-pulse test stimulation (TS; 1/30 Hz) on the afferent nerve evoked a single action potential, while repetitive stimulation (RS; 1 Hz) induced a long-lasting SRP that was abolished by a selective Ca(2+)/CaMKII inhibitor, autocamtide 2-related inhibitory peptide (AIP). Such an inhibitory effect was reversed by a relative excess of nitric oxide synthase (NOS) substrate, L-arginine. In addition, the RS-induced SRP was abolished by pretreatment with the NOS inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME). The sGC activator, protoporphyrin IX (PPIX), reversed the blocking effect caused by L-NAME. On the other hand, a sGC blocker, 1H-[1, 2, 4]oxadiazolo[4, 3-alpha]quinoxalin-1-one (ODQ), abolished the RS-induced SRP. Intrathecal applications of the membrane-permeable cGMP analog, 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt monohydrate (8-Br-cGMP), reversed the blocking effect on the RS-induced SRP elicited by the ODQ. Our findings suggest that a CaMKII-dependent NO/sGC/PKG pathway is involved in the RS-induced SRP, which has pathological relevance to hyperalgesia and allodynia.     

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one induces cell cycle arrest and apoptosis in HeLa cells by preventing microtubule polymerization

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) is known as a specific inhibitor of soluble guanylyl cyclase (sGC). Previously, however, ODQ was reported to induce cell death via sGC-dependent and sGC-independent means in a variety of cell types. The aim of this study was to investigate the mechanism by which ODQ induces cell death in HeLa cells.Treatment of HeLa cells with ODQ induced a concentration-dependent decrease in cell viability over the range from 10 to 100 μM. DNA fragmentation and fluorescence-activated cell sorting analysis using annexin V and propidium iodide staining revealed that ODQ triggered apoptosis at concentrations of 50 and 100 μM within 24 to 48 h. The addition of 8-Br-cGMP in the presence of ODQ failed to rescue HeLa cells from death, suggesting that the inhibition of sGC was not responsible for the pro-apoptotic action of ODQ. ODQ arrested the cell cycle at the G2/M phase and caused disassembly of the microtubule network. This process was reversed by dithiothreitol. In addition, ODQ was shown to inhibit the polymerization of purified tubulin, and this was also prevented by dithiothreitol. These results indicate that ODQ inhibits microtubule assembly by direct oxidation of tubulin, induces cell cycle arrest at the G2/M phase, and triggers apoptosis in HeLa cells.     

Nebivolol-induced vasodilation of renal afferent arterioles involves β3-adrenergic receptor and nitric oxide synthase activation

Nebivolol is a β(1)-adrenergic blocker that also elicits renal vasodilation and increases the glomerular filtration rate (GFR). However, its direct actions on the renal microvasculature and vasodilator mechanism have not been established. We used the in vitro blood-perfused juxtamedullary nephron technique to determine the vasodilator effects of nebivolol and to test the hypothesis that nebivolol induces vasodilation of renal afferent arterioles via an nitric oxide synthase (NOS)/nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP pathway and the afferent arteriolar vasodilation effect may be mediated through the release of NO by activation of NOS via a β(3)-adrenoceptor-dependent mechanism. Juxtamedullary nephrons were superfused with nebivolol either alone or combined with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or the NOS inhibitor N(ω)-nitro-l-arginine (l-NNA) or the β-blockers metoprolol (β(1)), butoxamine (β(2)), and SR59230A (β(3)). Nebivolol (100 μmol/l) markedly increased afferent and efferent arteriolar diameters by 18.9 ± 3.0 and 15.8 ± 1.8%. Pretreatment with l-NNA (1,000 μmol/l) or ODQ (10 μmol/l) decreased afferent vasodilator diameters and prevented the vasodilator effects of nebivolol (2.0 ± 0.2 and 2.4 ± 0.6%). Metoprolol did not elicit significant changes in afferent vasodilator diameters and did not prevent the effects of nebivolol to vasodilate afferent arterioles. However, treatment with SR59230A, but not butoxamine, markedly attenuated the vasodilation responses to nebivolol. Using a monoclonal antibody to β(3)-receptors revealed predominant immunostaining on vascular and glomerular endothelial cells. These data indicate that nebivolol vasodilates both afferent and efferent arterioles and that the afferent vasodilator effect is via a mechanism that is independent of β(1)-receptors but is predominantly mediated via a NOS/NO/sGC/cGMP-dependent mechanisms initiated by activation of endothelial β(3)-receptors.     

Peroxynitrite can affect platelet responses by inhibiting energy production

Peroxynitrite (ONOO-) strongly inhibits agonist-induced platelet responses. However, the mechanisms involved are not completely defined. Using porcine platelets, we tested the hypothesis that ONOO- reduces platelet aggregation and dense granule secretion by inhibiting energy production. It was found that ONOO- (25-300 microM) inhibited collagen-induced dense granule secretion (IC50 = 55 +/- 7 microM) more strongly than aggregation (IC(50) = 124 +/- 16 microM). The antiaggregatory and antisecretory effects of ONOO- were only slightly (5-10%) reduced by 1H-[1,2,4]-oxadiazolo-[4,3-alpha]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. In resting platelets ONOO- (50-300 microM) enhanced glycolysis rate and reduced oxygen consumption, in a dose dependent manner. The ONOO- effects on glycolysis rate and oxygen consumption were not abolished by ODQ. The extent of glycolysis stimulation exerted by ONOO- was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or an uncoupler (2,4-dinitrophenol). Stimulation of platelets by collagen was associated with a rise in mitochondrial oxygen consumption, accelerated lactate production, and unchanged intracellular ATP content. In contrast to resting cells, in collagen-stimulated platelets, ONOO- (200 microM) distinctly decreased the cellular ATP content. The glycolytic activity and oxygen consumption of resting platelets were not affected by 8-bromoguanosine 3',5'-cyclic monophosphate. Blocking of the mitochondrial ATP production by antimycin A slightly reduced collagen-induced aggregation and strongly inhibited dense granule secretion. Treatment of platelets with ONOO- (50-300 microM) resulted in decreased activities of NADH : ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome oxidase. It is concluded that the inhibitory effect of ONOO- on platelet secretion and to a lesser extent on aggregation may be mediated, at least in part, by the reduction of mitochondrial energy production.